During the recent years, techniques have been developed for the genetic manipulation of plant cells and the regeneration of these plant cells into transgenic plants. On the one hand, direct DNA transformation of plant protoplasts may be used for the introduction of the desired DNA into plant cells. For this purpose, several methods are available, e.g. Ca/PEG (Krens et al., 1982; Negrutiu et al, 1987), electroporation and microinjection (Crossway et al., 1986). Using the recently developed microprojectile method (Klein et al, 1987) also intact plant tissues may be transformed with `naked` DNA. On the other hand, the desired DNA may be introduced into the plant cell using the natural DNA transfer system of Agrobacterium tumefaciens and Agrobacterium rhizogenes bacteria (for review, see Klee et al., 1987).
Agrobacterium tumefaciens and Agrobacterium rhizogenes, after attachment to the plant cell wall, are capable of transferring a piece of DNA to the plant cell. Such a piece, the transfer-DNA (T-DNA), is as T-region part of a large plasmid (190-240 kbp) in the bacterium, which is called the Ti-plasmid in the case of A. tumefaciens and Ri-plasmid in the case of A. rhizogenes. The T-DNA becomes integrated into the nuclear genome of the plant cell (Tomashow et al., 1980; Chilton et al., 1982). Genes residing in the T-DNA are expressed in the plant cell and cause the latter to behave as a tumor cell (Ooms et al., 1981; Willmitzer et al., 1982a+b).
In addition to the genes that are responsible for tumor induction also genes are present on the T-DNA which take care of the production of so-called opines. Opines, like octopine and nopaline, may serve as energy, nitrogen and/or carbon source to Agrobacterium. The enzymes that are needed for the catabolism of these opines are encoded by genes that reside on the Ti- (Ri-) plasmids (e.g. Bomhoff et al., 1976; Kerr and Roberts, 1976; Hooykaas et al., 1977). Depending on the opine production, the Ti- and Ri-plasmids are classified into groups (for example octopine or nopaline plasmids).
The T-region is confined by two imperfect direct repeats of 25 base pairs, also called `borders` (Yadav et al., 1982; Zambryski et al., 1982; Gielen et al., 1984; Slightom et al., 1985). The presence of these borders in cis is a prerequisite for correct transfer of T-DNA (Wang et al., 1984; Peralta and Ream, 1985).
The presence of the right border is necessary for the efficient T-DNA transfer (Ooms et al., 1982; Shaw et al., 1984b; Wang et al., 1984). Depending on the test system it was found that deletion of the left border in some experiments does (Bakkeren et al., 1989) whereas in other experiments does not lead to a lower frequency of T-DNA transfer to the plant cell (Hille et al., 1983a; Joos et al., 1983). Next to the right border a sequence is present that significantly increases the efficiency of T-DNA transfer (Peralta et al., 1986; Van Haaren et al., 1986, 1987; Wang et al., 1987). The action of this `enhancer` element is independent on position or orientation with respect to the right border (Van Haaren et al, 1986). From experiments with synthetic borders it appeared that the right and left border sequences are interchangeable and, consequently, the `enhancer` determines which border sequence becomes the dominant right border (Peralta et al., 1986; Van Haaren et al., 1987).
In addition to the T-DNA, there are virulence genes that on the one hand reside on the chromosome, on the other hand on the Ti-plasmid (Vir-region). These genes are involved in attachment of the bacterium to the plant cell and in the transfer process of the T-DNA to the plant cell (for review see Melchers and Hooykaas, 1987) .
All the gene transfer systems mentioned above have in common the disadvantage that the site of integration of the transforming DNA is unpredictable. Thus, as with the other plant transformation techniques mentioned above, the DNA that is introduced into the plant cell via Agrobacterium appears to become integrated at random locations in the genome (Chyi et al., 1986; Wallroth et al., 1986; Spielman and Simpson, 1986). In certain situations, however, it is desirable or even necessary to determine the site of integration beforehand. Thus, the gene to be introduced might be targeted to a location where the desired regulation of expression is guaranteed. Also the newly introduced DNA could be used to mutate or inactivate a specific plant gene. Several methods have been described to integrate DNA sequences into the plant genome in a site-specific manner. These methods are all based on a mechanism known as homologous recombination.
Homologous recombination is a process that occurs very efficiently within bacteria and yeasts. In these organisms it is used for site-directed integration of newly introduced DNA (Ruvkun and Ausubel, 1981; Orr-Weaver et al., 1981). In yeast it was found that DNA molecules, linearized in the area of homology with DNA integrated into the genome, recombine with a 10-1000 fold higher frequency. More recently, also in mammalian cells homologous recombination between genomic and newly introduced DNA was found to occur (Smithies et al., 1985; Thomas and Capecchi, 1987; Song et al., 1987; Baker et al., 1988; for recent review see Capecchi, 1989). Also in these systems it appeared that upon co-transformation of two defective mutants linearisation of one of the mutants in the region of homology resulted in--on an average--a 10-fold higher recombination frequency (Kucherlapati et al., 1984).
Recombination between two homologous DNA molecules, after their simultaneous introduction into a plant cell, has been reported by Wirtz et al., 1987. European patent application (EP-A-0 317 509) discloses a method for the integration of DNA sequences into the genome of plants through homologous recombination. According to the application the introduction of the DNA construct into the plant host may occur by known techniques, such as the Agrobacterium transfer system. In the Examples, a direct DNA transformation method (with "naked" DNA) was actually used to introduce the incoming DNA into polyethyleneglycol (PEG) treated tobacco protoplasts.
It was stated that modifications on exactly defined locations in the plant genome could be obtained. However, the results of the experiments, using different defective APHII genes, conferring kanamycin resistance, were not conclusive as to whether restoration of the gene occurred on the desired locus ("in situ"). In a later published article on the same experiments by one of the inventors, Paszkowski et al. (1988), it was only assumed that restoration of the defective APHII gene, due to homologous recombination with the incoming defective;APHII gene, could have occurred on locus, "but further evidence to confirm this was required".
There is still a need for an efficient method for in situ modification of the plant genome and selection of the desired mutants.